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Background of CBX4 / PC2 antibody

PC2 is the human homolog of the Drosophila 'Polycomb' (Pc) protein which has been identified as a gene family member associated with a cellular memory system that is responsible for the inheritance of gene activity by progeny cells. The human Pc homolog gene is more closely related to a Xenopus Pc homolog, XPc, than to a previously described human Pc homolog, CBX2 (hPc1). However, the hPc2 and CBX2/hPc1 proteins are shown to colocalize in interphase nuclei of human U-2 OS osteosarcoma cells, suggesting that the proteins are part of a common protein complex. The human protein is believed to function as a repressor of proto-oncogene activity and that interference with hPc2 function can lead to derepression of proto-oncogene transcription and subsequently to cellular transformation. Other reports describe PC2 as a protein that has SUMO E3 activity for the corepressors CtBP and CtBP2.

General readings

Simon JA and Tamkun JW. Programming off and on states in chromatin: mechanisms of Polycomb and trithorax group complexes. Curr. Opin. Genet. Dev. 2002;12:210-8.

Formalin-fixed and paraffin-embedded human cancer tissue reacted with CBX4 polyclonal antibody ( Cat # PAB1690 ) , which was peroxidase-conjugated to the secondary antibody, followed by AEC staining. This data demonstrates the use of this antibody for immunohistochemistry; clinical relevance has not been evaluated. HC = hepatocarcinoma.

Figure 2. Formalin-fixed and paraffin-embedded human cancer tissue reacted with the primary antibody, which was peroxidase-conjugated to the secondary antibody, followed by AEC staining. This data demonstrates the use of this antibody for immunohistochemistry; clinical relevance has not been evaluated.

Western blotting analysis shows the detection of human CBX4 in probed lysates using CBX4 polyclonal antibody ( Cat # PAB10228 ).The panel on the left shows the blot probed with CBX4 polyclonal antibody.The panel on the right is the same blot reprobed with anti-FLAG antibody to confirm the presence of FLAG tagged recombinant CBX4 in the lysate.In the left panel the band labeled as CBX4 is FLAG-tagged transfected CBX4 in 293T cells.The bands labeled with stars are likely endogenous CBX4.In the right panel the band labeled CBX4 is FLAG-tagged transfected CBX4 in 293T cells.Data contributed by Dr. Ari Melnick, Albert Einstein College of Medicine.

anti-hPC2 antibody was used for immunofluorescent imaging of human cells (U2OS). The image reveals the expected discrete nuclear structure that is termed the PcG body corresponding to the known localization of PC2 (see Satijn et al. below). IF was performed after fixation in PBS with 4% PF for 5 min, permeabilization with 0.5% Triton X100-PBS for 5 min, and blocking with 5% milk / 0.2% Tween for 1 h. Primary antibody used at 1:200 in 5% milk / 0.2% Tween for 1 h, secondary antibody for 30 min.

Determination of the antibody titer To determine the titer of the antibody, an ELISA was performed using a serial dilution of the antibody against human CBX4. The plates were coated with the peptides used for immunization of the rabbit. By plotting the absorbance against the antibody dilution (Figure 1), the titer of the antibody was estimated to be 1:11,250.